Iron participates in a wide variety of cellular reactions and is an essential element for nearly all organisms. Iron acquisition is problematic for pathogenic bacteria, which must compete with the host for limited amounts of available iron, and bacteria typically express high affinity transport systems that are specific for their ferric or ferous iron ligands. Feo is the major ferrous iron transport system in prokaryotic organisms. It is widely distributed in eubacteria and in the Archaea, suggesting that it is a very ancient class of iron transporter. Further, the FeoB protein has GTPase and GDI domains similar to eukaryotic G proteins, and Feo may represent a primitive ancestor of modern G proteins. Despite its ubiquitous nature and potential role in bacterial pathogenesis, little is known about its structure, mechanism of transport, and biological role. Thus, it is critical to characterize this protein and its role in ferrous iron transport. These studies will be done in the pathogen Vibrio cholerae, a major cause of human morbidity and mortality. V. cholerae is an ideal model for these studies as we have the reagents and genetic tools to answer basic questions about Feo. Further, we can extend these studies to include questions about the role of Feo in host colonization and disease, which will lay the groundwork for developing new methods of blocking infection by V. cholerae. Our first specific aim is to use genetic and biochemical approaches to characterize Feo structure and its mechanism of transport. These studies will help define the essential protein components of the Feo transporter and the critical regions and amino acids within each protein. Based on these results, we will be able to test models for Feo structure and function. Our second specific aim is to characterize the accessory ferrous iron transport protein, VciB. This may provide insight into how Feo obtains its ferrous iron ligand in the periplasm. Third, we will define the mechanism of regulation of expression of the feo operon and determine its pattern of expression when the bacteria are within the host. These data will provide new information about how pathogenic bacteria coordinate the expression of their iron transport genes to allow optimal utilization of available iron sources and will also help define the environmental signals the bacteria encounter in the host. PUBLIC HEALTH RELEVANCE: Obtaining essential iron from the host is a critical step in production of disease by bacterial pathogens. Characterizing the ferrous iron uptake system Feo in Vibrio cholerae will provide important information about an iron transporter found in many bacterial pathogens, and it will further our knowledge of the organism that causes cholera. Because no system comparable to Feo is found in the human, this iron transport system may represent a new class of targets for antimicrobial therapy.